An Experimental Study Of Biodegradable PBAE Nanoparticles Mediated HSVtk/GCV System For Rat Glioma Therapy

The biodegradable non-viral polymeric nanoparticles have great value in the field of gene therapy. Compared to other viral cargo carriers, polymeric nanoparticles are easier to synthetize, have characteristics with varied structures and have better and a higher gene delivery capacity. Nanoparticles also have fewer potential biosafety concerns as compared to viral cargos, which make polymeric nanoparticles great gene-delivery candidates in the development of clinically relevant gene therapy. However, according to the previous reports, polymeric nanoparticles have demonstrated weak tissue filtrating ability with low transfection rates. These issues have plagued the development of tumor gene therapy via polymeric nanoparticles gene delivery.In the first part of our study, we chose a newly developed biodegradable polymer type- poly(beta-amino ester)s- as the basic molecular structure of the polymer. After synthetizing and screening a series of polymers, we chose the optimal polymer based on stable physical & chemical characteristics and a high cell transfection rate. The candidate polymer 447 with base molecules 1,4-butanediol diacrylate(B4) and 4-amino-1-butanol(S4), and end capped with 1-(3-aminopropyl)-4-methylpiperazine(E7) was the optimal candidate. This PBAE 447 molecule binds with DNA molecules and self-assembles into particles of nanometer size. In this way, we assembled the p EGFP-N1(GFP) plasmid and herpes simplex virus type I thymidine kinase(HSVtk) plasmid with the 447 polymer. Therefore PBAE-GFP nanoparticles and PBAE-HSVtk nanoparticles were synthetized. The transfection test on two rodent glioma cell lines, F98 and 9L, demonstrated that the best mass ratios between polymer and DNA were 30 w/w.Introducing a “suicidal gene” into the tumor cells, which could induce tumor cell death, is a key strategy in tumor gene therapy. The Herpes simplex thymidine kinase/ganciclovir(HSVtk/GCV) system is the most widely used system in the field of gene therapy. GCV is a type of prodrug, and HSVtk phosphorylates GCV from its original form into the activated form, which can block DNA synthesis and induce tumor cell death.In the second part of our study, we evaluated the role of PBAE nanoparticles in mediating the HSVtk/GCV system to the transfect the tumor cells. By applying GCV to the cells, our results confirmed that our PBAE nanoparticles with HSVtk gene could kill F98 and 9L cells, while PBAE-GFP nanoparticles with no GCV showed no effect on the cells. Different from other studies on the HSVtk/GCV system, our results demonstrated that the tumor cells could be successfully transfected and the HSVtk/GCV system showed a significant cytotoxic effect on the cultured cells in vitro.With considerations regarding a more practical usage of the nanoparticles, we lyophilized the fresh synthesized nanoparticles. Based on our results, there was no significant difference between several forms of PBAE nanoparticles, including fresh prepared, lyophilized and Cy5 labeled nanoparticles. Their chemical & physical features remained stable, and there was no significant difference between cell transfection rates among the groups. Further studies on the transfection time-course demonstrated that the expression of a foreign gene could last for 4 days and more, and the peak time of expression was on Day 2.Another drawback for the non-viral nanoparticle cargo is the low filtrating ability in vivo. Polymeric nanoparticles are typically very difficult to get through the narrow interstitial space inside tissue. Therefore, the volume of distribution of chemotherapeutic drugs is typically very limited. Convection-enhanced delivery(CED) system is a novel drug delivery method. By applying and maintaining a low pressure gradient, CED can slowly infuse the drugs into the local tissue. Because the CED is more dependent on the hydrostatic pressure rather than liquid diffusing force, the capacity and volume of distribution of drugs inside the tumor entities can be much larger than any other infusing ways.In the third part of our study, we intracranially implanted syngeneic 9L glioma cell pieces into the cortex of rats. We then infused PBAE-HSVtk nanoparticles into the tumor cavity and delivered GCV through intraperitoneal(IP) injection. The rats were then observed daily and the treatment responses were recorded and analyzed. Our results showed that there was no adverse reaction from the PBAE nanoparticles to the rat brain tissue. It was safe and reliable to use PBAE nanoparticles as the cargo of gene therapy. By labeling the nanoparticles with Cy5, we tracked the location and distribution of PBAE nanoparticles inside the brain. We were able to show that almost all of the tumor tissue was transfected by the nanoparticles, including the needle position and border areas which were remote from the injection center. Additionally, survival analysis showed that the median survival of the group treated with PBAE-HSVtk nanoparticles was significantly longer than other groups(p = 0.0012).Altogether, this work presents a biodegradable nanomedicine approach capable of effectively and selectively delivering DNA to malignant glioma in vivo. A library of PBAE nanoparticles was evaluated in vitro to optimize DNA delivery while minimizing toxicity. The optimal nanoparticle formulation was then physically characterized. We demonstrate that these nanoparticles can deliver an HSVtk transgene in vitro and initiate glioma cell killing via the local activation of GCV. We also demonstrate transfection of malignant gliomas in vivo. Using this nanoparticle-based therapy, we were able to statistically improve the survival of rats with malignant glioma. This work presents an exciting frontier in nanomedicine with significant potential to treat malignant gliomas.